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Fluoroquinolone Residues in Raw Cow's Milk

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Fluoroquinolone Residues in Raw Cow's Milk Czech J. Food Sci. Vol. 29, 2011, No. 6: 641–646 Fluoroquinolone Residues in Raw Cow’s Milk Pavlína NAVR ÁTILOVÁ, Ivana BORKOvcOVÁ, Jana VYHNÁLKOVÁ and Lenka VORLOVÁ Department of Milk Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic Abstract Navrátilová P., Borkovcová I., Vyhnálková J., Vorlová L. (2011): Fluoroquinolone residues in raw cow’s milk. Czech J. Food Sci., 29: 641–646. The aim of the study was to monitore the levels of fluoroquinolone residues in bulk samples of raw cow’s milk. The bulk samples of raw cow’s milk (n = 150) were obtained from 58 different milk suppliers in the South Moravian and Vysočina Regions. The samples were analysed by reverse phase high-performance liquid chromatography method with fluorescence detection and gradient elution. 87.3% of the raw milk samples were positive for the fluoroquinolones residues. Flumequine was present in none of the samples. The levels of other fluoroquinolones investigated were be- low the recommended maximum residue limit. The results of the study indicate that fluoroquinolones are frequently administered to the dairy cows in spite of the recommendations of CVMP. The most frequently determined was en- rofloxacin and its indicator residue, i.e. ciprofloxacin. An efficient control of the veterinary drugs residues in milk is very important to ensure the safety of the milk and milk products. Keywords: veterinaty drug; RP HPCL; food safety; MRL; LOD; LOQ The monitoring of the veterinary drugs residues bacterial agents in both human and veterinary is an important part of the food safety control in medicine. Fluoroquinolones are effective for the raw materials and foods of animal origin. To ensure therapy of serious diseases, e.g. septicaemia, gas- the safety of food of animal origin for consumers, troenteritis, and respiratory diseases caused by maximum residue limits (MRL) of veterinary drugs Gram-negative bacteria. They are also used for used with the food animals have been set for raw the treatment of infections of the urinary tract materials of animal origin (Botsoglou & Fle- and skin, and of infections of soft tissues caused touris 2001; Commission Regulation 37/2010). by Gram-negative and certain Gram-positive aero- Since 1976, when the first monofluoroquinolone bic bacteria. They are effective in the therapy for flumequine was developed, very many fluoroqui- mycoplasma infections and infections caused by nolone representatives have been synthetised and atypical bacteria. Incidence of side adverse effects described. Compared with the classic 1st generation of fluoroquinolone treatment, particularly in hu- quinolones that were characterised by a narrow man medicine, has been reported (Flomenbaum spectrum of action and poor tissue penetration et al. 2006; CVMP 2007). ability, fluoroquinolones feature many attributes In veterinary medicine, they are useful especially of ideal antimicrobials (Sarkőzy 2001; Emmer- in the therapy for gastrointestinal and respiration son & Jones 2003). Fluoroquinolones are used infections (Botsoglou & Fletouris 2001). Fluo- for the treatment of infections caused by various roquinolones are used in intramammary prepara- Supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project No. MSM 6215712402. 641 Vol. 29, 2011, No. 6: 641–646 Czech J. Food Sci. tions for the treatment of mastitis in lactating dairy enrofloxacin (ENRO) and its indicator residue = cows, for dairy cows during dry periods, and also sum of enrofloxacin and ciprofloxacin (CIPRO), for mastitis prevention (Gruet et al. 2001). and the residues of marbofloxacin (MARBO), Although fluoroquinolones found a wide applica- danofloxacin (DANO) and flumequine (FLU), and tion in primary agriculture, their administration to to determine their concentrations. food animals has been discussed in recent years in connection with the increased incidence of resist- ance among human pathogenic micro-organisms. MATERIAL AND METHODS From the onset of the bacterial resistance point of view, fluoroquinolones belong to the high-risk Milk samples. The bulk samples of raw cow’s groups of antimicrobial drugs (WHO 1998; CVMP milk (n = 150) were obtained from 58 different 2007). The World Health Organisation (WHO) milk suppliers in the South Moravian and Vysočina and Word Organisation of Animal Health (OIE) Regions. The samples of bulk milk were collected have defined quinolones as “critically important from the milk collection route of the dairy plant. antimicrobials” for human and animal health, The milk samples were kept at temperatures not respectively (FAO 2007). exceeding 4°C until the testing time. Due to extra-label use of veterinary drugs or Chemicals and materials. The standards of fluo- noncompliance withdrawal periods, much higher roquinolones, i.e. of enrofloxacin (17849), flume- residue levels might appear in the edible animal quine (F7016), marbofloxacin (34039), danofloxacin products (Botsoglou & Fletouris 2001). The (33700), ciprofloxacin (17850), and norfloxacin published data indicated that violative residues (N 9890) were purchased from Sigma Aldrich (St. were found in raw or heat treated milk samples in Louis, USA). The chemicals for HPLC analysis, i.e. countries all over the world (Botsoglou & Fle- acetonitril, methanol and dichloromethane (HPLC touris 2001; Tolentino et al. 2005; Fonseca et grade) were obtained from Merck (Darmstadt, al. 2009; Kaya & Filazi 2010; Addo et al. 2011). Germany). Trifluoroacetic acid and phosphoric Quality control of the purchased raw cow’s milk acid (suprapure grade) were from Fluka Chemie implies that 0.16% samples showed violative con- AG (Buchs, Switzerland) and Sigma Aldrich (St. centrations of residues (Kopunetz 2010). Louis, USA). NaOH (analytical grade), was pur- Within the EU, each state is obliged to monitor chased from Penta (Prague, Czech Republic). The foodstuffs for the residues of veterinary drugs hardware included an analytical balance (Kern & and to present a National Residue Monitoring Sohn GmbH, Balingen, Germany), a cooling cen- Plan that takes into account the specific situa- trifuge (Mechanika Precyzyjna, Bytom, Poland), tion in its country (Botsoglou & Fletouris a rotary vacuum evaporator (Bűchi, Postfach, 2001). In the Czech Republic, milk is monitored Switzerland), and a vortex (Merck, Darmstadt, for the presence of veterinary drugs residues in Germany). accredited laboratories as part of their food chain Preparation of standard fluoroquinolone so- monitoring for exogenous substances, and also lutions. The stock solutions at active substance during quality checks of the raw milk. Screening concentration of c = 1 g/l were first prepared by tests for veterinary drugs residues in milk rely dissolving an adequate amount of the chemothera- mainly on rapid tests and microbial inhibition peutic in 1 ml of 0.1 mol/l NaOH and adding deion- methods (plate diffusion method) (Botsoglou ised water to a total volume of 25 ml. The stock & Fletouris 2001; Czech National Reference solutions of chemotherapeutics were then further Laboratory 2008). In targeted testing for fluoroqui- diluted with deionised water to obtain working nolone residues, modern analytical methods such solutions with a concentration in the 0.1–1.0 mg/l as the high-performance liquid chromatography range that were then used to prepare fortified milk (HPLC) analysis and mass spectrometry are used samples for the method validation. (Hernández-Arteseros et al. 2002; Marazuela Preparation of samples for HPLC analysis. & Moreno-Bondi 2004). Quinolones were extracted from the milk sam- The objective of the present study was to give the ples by the liquid/liquid extraction method. Con- information on the presence of fluoroquinolone centrated phosphoric acid solution (0.5 ml) was residues for which maximum residue limits were added to 5 ml of milk and vortexed for 5 seconds. set in raw cow’s milk. These imply the residues of Acetonitrile (10 ml) was pipetted into the mixture 642 Czech J. Food Sci. Vol. 29, 2011, No. 6: 641–646 for extraction by vortexing for 1 minute. The re- marbofloxacin, 10.0–400.0 μg/l for norfloxacin, sulting mixture was then centrifuged at 4000 rpm 6.5–260 μg/l for ciprofloxacin, 2.0–80.0 μg/l for for 10 min at 5°C. The supernatant was collected danofloxacin, 6.2–250.0 μg/l for enrofloxacin, and (10 ml) and acetonitrile was evaporated. Two ml 1.2–48.0 μg/l for flumequine. of dichloromethane were added to the residue, The limit of detection (LOD) and the limit of and the mixture was again vortexed for 10 s and quantification (LOQ) were determined from the centrifuged at 4000 rpm for 10 min at 5°C. The analysis of the matrix samples fortified with fluo- 1 ml sample collected from the upper aqueous roquinolone standards, and evaluated as 3 signal layer was diluted with the mobile A phase at a to noise ratio (S/N) and 10 S/N in µg/kg. The re- ratio of 1:1 for HPLC analysis. peatability and recovery were determined from 12 HPLC conditions. The HPLC assay was per- parallel analyses of the milk samples fortified with formed on an Alliance 2996 liquid chromatograph the standards solutions of known concentrations, with a 2475 fluorescence detector (Waters, Mil- 0.5, 1.0, and 1.5 MRL. To determine the analyte ford, USA). The separation was performed on recovery for the individual fluoroquinolones, the an Atlantis T3 chromatographic column 4.6 × analytical results obtained with the fortified sam- 250 mm with the particle size of 5 μm (Waters, ples were compared with an externally generated Milford, USA). The mobile A phase consisted of calibration curve of the standards. Their mean 0.2% trifluoroacetic acid, the mobile B phase of a values are summarised in Table 1 together with methanol + acetonitrile mixture (1:5), the gradi- the coefficients of variation for repeatability. The ent was linear from 25% B phase to 80% B phase, decision limit CCα and the detection capability and the flow rate was 0.7 ml/min. The injection CCβ as defined by the Commission Decision 657 volume was 20 μl and the column temperature (2002) were determined by analysing the matrix was 30°C.
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